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Interferences in the Optimization of the MTT Assay for Viability Estimation of Proteus mirabilis.

Avicenna journal of medical biotechnology (2015-11-26)
Ewa Grela, Adam Ząbek, Agnieszka Grabowiecka
ABSTRACT

The chromogenic assay based on MTT bioreduction was adapted to Proteus mirabilis viability estimations. We primarily intended to use the assay for the evaluation of novel antimicrobial compounds, including structures with possible permeabilizing activity. Therefore, the influence of basic permeabilizing agents like Triton X-100 and EDTA upon the MTT assay was studied. 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) was used as a substrate for the whole-cell dehydrogenase activity estimations. The amount of formazan product was evaluated in the end-point reactions terminated with acidic isopropanol or in the continuous reactions run in the presence of low detergent concentrations. The generally established procedure of the end product dissolution with acidic isopropanol caused absorbance instability which strongly affected the results accuracy. The disadvantage was especially pronounced when the assay was conducted in Mueller-Hinton Broth. PBS with 0.01% Triton X-100 used as the reaction medium allowed to omit the formazan dissolution step and follow the microbial MTT reduction in a continuous mode. It was observed that in Proteus mirabilis with a compromised outer membrane the assay score was artificially increased above the untreated control. The dependence of the assay results on the cell integrity might be a major drawback of the MTT assay application for the evaluation of novel antimicrobials against Gram-negative microorganisms. On the other hand, the MTT reduction could be conveniently used to assay the permeabilization degree in biotechnological protocols.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Triton X-100, laboratory grade
Sigma-Aldrich
Thiazolyl Blue Tetrazolium Bromide, powder, BioReagent, suitable for cell culture, suitable for insect cell culture, ≥97.5% (HPLC)
Sigma-Aldrich
Thiazolyl Blue Tetrazolium Bromide, 98%
Sigma-Aldrich
Ethylenediaminetetraacetic acid, ACS reagent, 99.4-100.6%, powder
Sigma-Aldrich
Ethylenediaminetetraacetic acid, BioUltra, anhydrous, ≥99% (titration)
Sigma-Aldrich
Ethylenediaminetetraacetic acid, anhydrous, crystalline, BioReagent, suitable for cell culture
Sigma-Aldrich
MTT Formazan, powder
Sigma-Aldrich
Ethylenediaminetetraacetic acid solution, 0.02% in DPBS (0.5 mM), sterile-filtered, BioReagent, suitable for cell culture
Sigma-Aldrich
Ethylenediaminetetraacetic acid, purified grade, ≥98.5%, powder
Sigma-Aldrich
Ethylenediaminetetraacetic acid, 99.995% trace metals basis
Sigma-Aldrich
MTT Formazan, MTT reduction end product